PROJECT SUMMARY Immunotherapy has been celebrated as a breakthrough cancer treatment, but despite this success, most patients fail to respond to treatment. Growing evidence indicates that the intestinal microbiota can contribute to responsiveness to immunotherapy. The gut represents a key site for immune regulation, and the delicate balance between immune surveillance and tolerance can fluctuate in response to changes in the microbiota, subsequently influencing systemic immunity and affecting extra-intestinal diseases (21). We and others have found that colonization with specific bacterial species in mice was sufficient to enhance anti-tumor immunity and augment immunotherapy (2, 22). We observed that oral administration of a Bifidobacterium cocktail alone was sufficient to improve tumor control and enhanced the efficacy of immunotherapy anti-PD-L1 (2). The benefit derived from Bifidobacterium appears to be connected to augmented dendritic cell function and CD8+ T cell priming. While this prior study identified key characters orchestrating this microbiota-based effect, the link connecting specific commensals in the gut to improved tumor control at distant sites is unclear, and whether extensions can be made to human health and cancer patients was previously unknown. My objective during my Ph.D. is to determine whether we can observe similar phenomenon in cancer patients, and to identify how intestinal commensals distantly affect the tumor microenvironment. Consistent with our preclinical data, we and others have found certain taxa associated with response to immune checkpoint blockade in cancer patients (1, 19, 20). Provocatively, we also observed that germ-free mice colonized with responder patient fecal material exhibited more robust anti-tumor immunity and derived greater benefit from anti- PD-L1 therapy compared to mice colonized by a non-responder. These data support a causal role for the gut microbiota in improved immunotherapy efficacy. I hypothesize that microbiota-dependent differences in anti- tumor immunity can be attributed to 1) microbially-derived or -induced circulating factors, 2) cells under local microbial influence at the intestine that migrate to the tumor, or 3) a combination of systemic and cellular factors. Methods to engineer model bacterial systems are rapidly evolving (13-16). These tools enable us to test hypothesis generated from mining databases with patient stool metagenomics data. The microbiome of cancer patients has not yet been interrogated to discover microbial compounds linked to patient response. In my post- doctoral work, I plan to focus on computational and synthetic biology approaches to find bacterial biosynthetic gene clusters and evaluate these gene products anti-tumor immune potentiating properties. I hypothesize that gene clusters overrepresented in the microbiome of patients that respond to immunotherapy can be expressed in heterologous bacterial systems to identify anti-cancer or immune-potentiating small molecules. My overarching goal is to continue building on the advances in this rapidly advancing field of the microbiome and cancer that will translate into clinical action.